Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36185
DC FieldValueLanguage
dc.contributor林納生zh_TW
dc.contributor蔡慶修zh_TW
dc.contributor孟孟孝zh_TW
dc.contributor陳浩仁zh_TW
dc.contributor.advisor徐堯煇zh_TW
dc.contributor.author黃纓雯zh_TW
dc.contributor.authorHuang, Ying-Wenen_US
dc.contributor.other中興大學zh_TW
dc.date2009zh_TW
dc.date.accessioned2014-06-06T07:54:08Z-
dc.date.available2014-06-06T07:54:08Z-
dc.identifierU0005-2307200818022600zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/36185-
dc.description.abstract竹嵌紋病毒衛星核酸(satBaMV RNA)是potexvirus病毒群中唯一發現的衛星核酸,為單股正極性的RNA分子,全長除了poly(A)外有836個核苷酸,其序列和竹嵌紋病毒(BaMV)之間並無顯著的同源性,但需要完全依賴BaMV進行複製、包被以及寄主細胞之間的散佈。在感染BaMV的煙草葉片中純化出複製的複合體,以建立分析satBaMV RNA 複製機制的生體外系統。結果發現由BaMV所組成的複製複合體不但可以辨識正確起始位置之正股、負股的satBaMV RNA為模板合成其全長之互補股,而且可以完成完整的satBaMV RNA複製循環,說明了satBaMV RNA和BaMV可能是利用同樣的複合體以進行核酸的合成。接著利用相同的方法抽取出狐尾草嵌紋病毒(FoMV)和馬鈴薯病毒X(PVX)的複製複合體,發現此兩種同屬於potexvirus的病毒亦可在生體外協助satBaMV RNA合成,然而在生體內的實驗中除了BaMV外只有FoMV可以輔助satBaMV RNA進行複製,包被以及移動,但其satBaMV RNA的累積量低於BaMV的協助。進一步的研究發現雖然FoMV可以協助satBaMV RNA複製但其包被satBaMV RNA的效率遠低於以BaMV為協助病毒導致satBaMV RNA在細胞內的不穩定而被降解。FoMV可感染至少56 種禾本科植物以及35 種雙子葉植物,此研究顯示衛星核酸之輔助病毒範圍可擴展至其他非同源的病毒進而有機會拓展satBaMV RNA 的寄主感染範圍。satBaMV RNA 3′端非轉譯區包含有複製所必須的因子:1.在satBaMV RNA 3′端非轉譯區所形成的二級結構中包含有三個stem-loops。2.與potexvirus病毒群相似的保留序列ACCUAA和AAUAAA。將其stem-loop破壞或刪除重要保留區的序列皆會嚴重的影響satBaMV RNA在細胞內的累積,這些因子在自然界中分離到的66種衛星核酸也有發現到,得知其在satBaMV RNA演化過程中是高度保留的,所以對於satBaMV RNA的生活史扮演重要的角色。satBaMV和BaMV的3′端非轉譯區具有相似的結構,除了BaMV比satBaMV多了一個偽結的三級結構,此結構可以和複製蛋白結合以作為合成負股RNA起始的辨識區,取代或置換此結構至satBaMV的3′端非轉譯區反而抑制了satBaMV RNA的合成,結果說明複製的複合體在辨識BaMV及satBaMV上的機制是不同的,有可能是關於參與其中的寄主因子是不同的。zh_TW
dc.description.abstractBamboo mosaic virus satellite RNA (satBaMV RNA) is the only example of satellite RNA present in the potexvirus group. It is a single-stranded RNA molecular of 836 nucleotide (nt) (excluding the 3' poly(A) tail) flanked by 159 nt of the 5'-untranslated region (UTR) and 125 nt of the 3'-UTR and shares no significant homology with BaMV RNA. SatBaMV RNA depends on BaMV for replication, encapsidation, and spread between the host cells. In vitro replication assays of satBaMV RNA were performed with RNA-dependent RNA polymerase (RdRp) complexes isolated from BaMV-infected Nicotiana benthamiana. The BaMV RdRp complexes not only correctly recognize promoter sequence of full-length positive- (+) or negative- (-) strand satBaMV RNA transcript for synthesis of complementary RNA but also complete the satBaMV RNA replication cycle. This revealed that the satBaMV and BaMV might use the same replication complexes for RNA synthesis. The same procedure was used for partial purification of Foxtail mosaic virus (FoMV) and Potato virus X (PVX) RdRp complexes and then for analysis of satBaMV RNA synthesis in vitro. Both of them could catalyze (+)- and (-)- satBaMV RNA for complementary RNA synthesis. Nevertheless, the in vivo inoculation tests revealed that besides BaMV, the replication, encapsidation, and movement of satBaMV were only supported by FoMV; however, the RNA accumulation efficiency is much lower than that by BaMV supporting. One possibility was that the ability of encapsidation of satBaMV RNA by FoMV coat protein was not sufficient for stabilizing satBaMV RNA in cells. FoMV infects at least 56 plant species in the Gramineae as well as a number of species in 35 dicotyledonous families. The satellite-supporting ability of FoMV was exploited in an attempt to extend the host range of satBaMV as a vector system. The 3'-UTR of satBaMV contains the cis-acting elements for replication: 1. secondary structure of satBaMV 3'-UTR comprises three stem-loops, design as SLA, SLB, and SLC. 2. conserved hexamer motif, ACCUAA, and polyadenylation signal AAUAAA. Disruptions of the secondary structures or deletions of the conserved sequences significantly decrease satBaMV RNA accumulation in plants. Analysis of natural satBaMV isolates revealed the conservation of the 3'-UTR structures which are crucial in the life cycle of satBaMV. BaMV and satBaMV 3'-UTRs share similar secondary structures and conserved elements; however, satBaMV 3'-UTR dose not contain the pseudoknot structure, which locates in the BaMV 3'-terminus and is required for BaMV RdRp binding and replication. Replacement of the pseudoknot structure or whole 3'-UTR of BaMV to satBaMV 3'-UTR interfered with the function of satBaMV RNA replication, implying that the recognition motif in the 3'-UTR and the replication mechanism of satBaMV might be different from that of its helper, BaMV RNA, and perhaps involve different host factors specifically for BaMV and satBaMV RNA replication.en_US
dc.description.tableofcontentsChinese abstract....i English abstract....iii Previous studies Bamboo mosaic virus....1 Satellite RNA of BaMV....2 Replication of BaMV....4 Replication of satBaMV RNA....5 Correlations between satellite RNAs, helper viruses, and host plants....5 References of previous studies....8 Objective....15 Chapter I. In vitro replication of Bamboo mosaic virus satellite RNA....16 Abstract....17 Introduction....18 Materials and methods Virus isolates and purification....20 BaMV RdRp preparation....21 Preparation of exogenous templates for RdRp assays....21 RdRp activity assay and analysis of products....23 Northern blot analysis....23 Rapid amplification of cDNA 5' ends (5' RACE) and DNA sequencing....23 Results Characterization of satBaMV RNA synthesized in vitro by BaMV RdRp preparation....24 Analyses of template activity of full-length satBaMV RNAs of different polarity....25 Analysis of the capability of BaMV RdRp preparation to complete the replication cycle of satBaMV RNAs in vitro...26 Specific internal initiation of (-)-sense satBaMV RNA by BaMV RdRp....28 Template activities of 5' UTR mutants of satBaMV....29 Discussion....30 References....36 Table....43 Figures....44 Chapter II. A non-cognate virus FoMV supports the replication, encapsidation, and movement of Bamboo mosaic virus satellite RNA in protoplasts and plants....50 Abstract....51 Introduction....52 Materials and methods FoMV and PVX RdRp preparation and RdRp assay....54 Virus purification and RNA extraction....54 SatBaMV transgenic N. benthamiana (line 2-6)....54 Synthesis of RNA transcripts in vitro....55 Protoplasts isolation, inoculation and plant infection...55 Total protein extraction and western blot analysis....55 Total RNA extraction and northern blot analysis....55 Grafting assays....57 Results and Discussions Synthesis of (+)- and (-)-satBaMV RNA in vitro by FoMV and PVX RdRp Preparations....57 FoMV induces the satBaMV RNA accumulation in the protoplasts of satBaMV transgenic N. benthamiana (line 2-6)....58 PVX, CyMV, and WClMV can not induce the satBaMV RNA accumulation in line 2-6 plants....59 FoMV supports the replication of satBaMV in line 2-6 protoplasts and plants....60 SatBaMV RNA was detected in the purified virion from FoMV infected line 2-6....61 SatBaMV RNA was moved from line 2-6 stock to non-transgenic scion by FoMV....62 References....64 Figures....68 Chapter III. Structural and functional analyses of the 3' untranslated region of satellite RNA of Bamboo mosaic virus....75 Abstract....76 Introduction....77 Materials and methods BaMV and satBaMV isolates....79 Preparation of 5'-end-labeled RNA for structural probing....79 Structural prediction and probing with ribonucleases....80 Characterization of the poly(A) tail of satBaMV RNA....81 Construction of deletion and substitution mutants of satBaMV....81 Synthesis of RNA transcripts in vitro....83 Protoplasts isolation, inoculation and plant infection...83 RNA analysis by northern blotting....84 Progeny RNA analysis....84 Linear comparison of RNA secondary structure....85 Results Secondary structure of the 3'-UTR of satBaMV RNA....86 Enzymatic probing of the secondary structure....86 Deletion analyses of the 3'-UTR for satBaMV RNA replication....87 Mutational analyses of SLA, SLB, and SLC for satBaMV RNA replication....89 Mutational analyses of conserved hexanucleotides for satBaMV RNA replication....90 Analysis of the interchangeability of the pseudoknot structure, 3'-UTR, and/or 5'-UTR between BaMV and satBaMV....91 Comparison of 3'-UTR secondary structures among BSF4 and natural occurring satBaMV isolates....92 Discussion....93 Sequence or structural elements in 3'-UTR play key roles in the replication and accumulation of viruses and subviral RNAs....93 Sequence elements heavily involved in the replication and accumulation in satBaMV 3'-UTR....95 Comparison of the roles of elements in 3'-UTR of BaMV and satBaMV....96 SatBaMV might have evolved a structurally similar but functionally distinct 3'-UTR for its efficient replication and accumulation....98 References....100 Tables....109 Figures....112 Publication....123en_US
dc.language.isoen_USzh_TW
dc.publisher生物科技學研究所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2307200818022600en_US
dc.subjectBamboo mosaic virusen_US
dc.subject竹嵌紋病毒zh_TW
dc.subjectsatellite RNAen_US
dc.subjectreplicationen_US
dc.subject衛星核酸zh_TW
dc.subject複製zh_TW
dc.title竹嵌紋病毒衛星核酸複製機制的研究zh_TW
dc.titleMechanism of Bamboo mosaic virus satellite RNA replicationen_US
dc.typeThesis and Dissertationzh_TW
item.languageiso639-1en_US-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.grantfulltextnone-
item.fulltextno fulltext-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
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